Shot-to-shot sampling using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer

ABSTRACT

Embodiments relate to an apparatus, method, or computer program. A laser may be configured to irradiate a plurality of laser pulses on a target area to ionize a sample placed in the target area into at least one ionized particle. Electrodes at a first end of a flight tube may be configured to accelerate ionized particles into the flight tube. A detector at a second opposite end of the flight tube may independently measure a time of flight of the ionized particles through the flight tube and an intensity of the ionized particles.

The present application claims priority to U.S. Provisional PatentApplication No. 62/377,768 filed on Aug. 22, 2016, which is herebyincorporated by reference in its entirety.

BACKGROUND

Data reproducibility in a measuring process of a Matrix-Assisted LaserDesorption/Ionization Time-Of-Flight Mass Spectrometer (MALDI-TOF MS) isan important concern in the design of a system for particularapplications. Since MALDI-TOF uses a solidified sample and matrixmixture on a plate onto which a laser is irradiated, it is inevitable tohave a non-uniform concentration or amount of sample mixture for eacharea of irradiation. The changes in the concentration or amount of asample as well as in structural formation of comprised molecules of asample at each laser irradiation may negatively influence thereproducibility. Measuring processes employ a summation or averagingprocess for the intensities acquired by laser shots, which yields thesummed or averaged intensities (e.g. from 500 to 1000 laser shots);unfortunately such a measuring process may have too highirreproducibility to be effective in certain applications (e.g. diseasediagnosis).

SUMMARY

Embodiments relate to an apparatus, method, or computer program. A lasermay be configured to irradiate a plurality of laser pulses on a targetarea to ionize a sample placed in the target area into at least oneionized particle. Electrodes at a first end of a flight tube may beconfigured to accelerate ionized particles into the flight tube. Adetector at a second opposite end of the flight tube may independentlymeasure a time of flight of the ionized particles through the flighttube and an intensity of the ionized particles.

In embodiments, independently measuring includes measuring adistribution of the time of flight and intensity of the at least oneionized particle. In embodiments, independently measuring includesindependently measuring the time of flight and the intensity of theionized particles for each pulse of the plurality of laser pulses.Independently measuring may compensate for physical variations in thesample, in accordance with embodiments. Independently measuring mayoptimize data reproducibility, in accordance with embodiments.Independently measuring may include independently measuring the time offlight and the intensity of the ionized particles for each locus of thesample.

In embodiments, a detector may output data which represents independentmeasurements. The data output may be compared to a reference library ofmass spectrometer reference data (e.g. for disease diagnosis). The dataoutput may be manipulated and/or analyzed prior to being compared to thereference library, in accordance with embodiments. In embodiments,manipulating and/or analyzing independent measurements includesimplementation of artificial intelligence and/or a deep learningalgorithm.

DRAWINGS

Example FIG. 1 illustrates a disease diagnosis laboratory, in accordancewith embodiments.

Example FIG. 2 is a schematic view of a MALDI-TOF MS system, inaccordance with embodiments.

Example FIG. 3 is a system diagram of the integrated system including asample processing unit, a MALDI-TOF MS unit, and a diagnosis unit in onesystem, in accordance with embodiments.

Example FIG. 4 is a system diagram of an integrated diagnostic systemincluding a sample processing unit and a MALDI-TOF MS unit integrated inone system, whereas a diagnosis unit is provided as a separate unit, inaccordance with embodiments.

Examples FIG. 5 illustrates a MALDI Plate where a spot on a sample plateis irradiated by a laser pulse, in accordance with embodiments.

Example FIG. 6 is a MALDI-TOF MS hardware diagram, in accordance withembodiments.

Example FIG. 7 illustrates a laser pulse applied to sample in MALDIchamber, in accordance with embodiments.

Example FIG. 8 illustrates an ionization of sample particles as a resultof laser exposure, in accordance with embodiments.

FIG. 9 is an example MALDI TOF mass spectra of ZnO, in accordance withembodiments.

FIG. 10 illustrates enlarged example mass spectra of FIG. 9 around 576m/z, in accordance with embodiments.

FIG. 11 is an example MALDI-TOF mass spectra, in accordance withembodiments.

FIG. 12 illustrates example peaks around 515 m/z corresponding to AuClparticles close to independent peaks corresponding to Zn particles ofFIG. 11, in accordance with embodiments.

DESCRIPTION

In mass spectrometry, matrix-assisted laser desorption/ionization(MALDI) is an ionization technique that uses a laser energy absorbingmatrix to create ions from large molecules with minimal fragmentation.It has been applied to the analysis of biomolecules (biopolymers such asDNA, proteins, peptides and sugars) and large organic molecules (such aspolymers, dendrimers and other macromolecules).

MALDI methodology may be a multi step process, in accordance withembodiments. First, a sample may be mixed with a suitable matrixmaterial and applied to a metal plate. Second, a pulsed laser mayirradiate the sample, triggering ionization and desorption of the sampleand matrix material. The sample molecules may be ionized by beingprotonated or deprotonated in the hot plume of ablated gases, and canthen be accelerated into a mass spectrometer (e.g. MALDI-TOF MS).

Embodiments relate to an apparatus, method, or computer program. A lasermay be configured to irradiate a plurality of laser pulses on a targetarea to ionize a sample placed in the target area into at least oneionized particle. Electrodes at a first end of a flight tube may beconfigured to accelerate ionized particles into the flight tube. Adetector at a second opposite end of the flight tube may independentlymeasure a time of flight of the ionized particles through the flighttube and an intensity of the ionized particles.

In embodiments, the sample comprises biological molecules.Characteristic information of the source may include a biologicalanalysis information of the source. The biological analysis informationmay be a medical diagnosis of either a human being, an animal, a plant,and/or a living organism.

Example FIG. 1 illustrates a disease diagnosis laboratory where a sampleprocessing facility 101 includes multiple sample processing tools, aMALDI-TOF MS system 102, and a diagnosis software system 103, which areseparated from each other, in accordance with embodiments. To extract aglycan for an ovarian cancer diagnosis, for example, a patient's serumis entered into a multi-well plate 111 to undergo a sample receptionprocess and a protein denaturation process 112, followed by adeglycosylation process using enzyme 113. A protein removal process 114,a drying and centrifugation process, a glycan extraction process 115,and a spotting process 116 then follow. The spotted samples are analyzedby the MALDI-TOF MS system 102 to generate at least one glycan profile.The diagnosis software 103 compares the glycan profile of the samplewith the pre-stored glycan profile or profiles to identity the presenceand progress of ovarian cancer. Example FIG. 1 is a schematic view of aMALDI-TOF MS system, in accordance with embodiments.

Example FIG. 3 is a system diagram of the integrated system including asample processing unit, a MALDI-TOF MS unit, and a diagnosis unit in onesystem, in accordance with embodiments. Samples may undergo acombination of process by selected modules. In the sample preparationsystem 301, a sample goes through a predefined and preprogrammedsequence depending on diagnosis or screening purposes in an automaticsample preparation unit 311. In embodiments, for glycan extraction,multiple processing modules may be selected, which as sample reception,protein denaturation, deglycosylation, protein removal, drying,centrifugation, solid phase extraction, and/or spotting. After samplepreparation, the sample loader 312 loads the samples onto the plates 306and are dried in a sample dryer 307.

The samples may then be provided to the MALDI-TOF MS unit 302 having aion flight chamber 321 and/or a high voltage vacuum generator 322, inaccordance with embodiments. A processing unit 323 in the MALDI-TOF MSmay identify the mass/charge (m/z) and its corresponding intensity. Forthe disease diagnostic purpose, those acquired mass and intensity datamay be reorganized to set up a standard mass list, in which a concept ofthe center of mass where intensities are balanced and equilibrated isintroduced. A standard mass to charge list is defined based upon themachine accuracy and the center of mass concept. The stored spectrumdata for each laser irradiation may also be used to set up the standardmass list. The diagnostic unit 303 may then compare, the spectra from apatient's sample with the pre-stored spectra and analyzes the patterndifference of the two spectra. The diagnostic unit may then identify thepresence and progress of the disease.

Example FIG. 4 is a system diagram of an integrated diagnostic systemincluding a sample processing unit and a MALDI-TOF MS unit integrated inone system, whereas a diagnosis unit is provided as a separate unit, inaccordance with embodiments. Example FIG. 4 illustrates an integrateddisease diagnosis system where the sample preparation unit 401 and theMALDI-TOF 402 are integrated, with the diagnosis unit 403 stands apartas a separate unit, in accordance with embodiments.

In embodiments, a detector outputs data which represents the independentmeasurements. The data output from the detector may be compared to areference library of mass spectrometer reference data. The samplereference library may be stored on a storage device, a Matrix-AssistedLaser Desorption/Ionization Time-of-Flight Mass Spectrometer (MALDI-TOFMS), a data storage device in the apparatus, a data storage deviceoutside the apparatus, a data storage device in communication with theapparatus through a network, a cloud storage system, a data storagedevice in communication with the apparatus through an internetconnection, and/or any other storage device or equivalent appreciated bythose skilled in the art.

In embodiments, data output from a detector is at least one ofmanipulated and/or analyzed prior to being compared to the referencelibrary. Manipulated and/or analysis may include implementation ofartificial intelligence and/or a deep learning algorithm. Inembodiments, manipulated and/or analyzed data may include calibrateddata produced by statistically analyzing the data output from adetector.

A calibration unit may be configured to statistically calibrate the dataoutput from the detector to maximize the reproducibility of the data, inaccordance with embodiments. In embodiments, the calibrating unit may beconfigured to calibrate intensities of the data output from the detectorby eliminating outliers of the intensity data for each mass-to-charge(m/z) peak associated with ionized particles. The calibrating unit maybe configured to select data sets including the data output from thedetector which shows a relative standard deviation (RSD) satisfying atriggering threshold. In embodiments, the triggering threshold may be apredetermined threshold, a dynamic threshold, a statistically determinedthreshold, a real-time varying threshold, a threshold determined byartificial intelligence, and or a threshold determined by a deeplearning algorithm.

In embodiments, the calibrating unit may be configured to select datasets from the data output from the detector utilizing continuous datadistribution analysis after the plurality of laser pulses. Inembodiments, selecting the data sets may use an algorithm by which apercentage of the data is eliminated. The algorithm may be performedbefore combining a plurality of data sets of the at least one data set,in accordance with embodiments. In embodiments, the combining mayinclude averaging and/or summing of the intensities of a plurality ofdata sets. In embodiments, a percentage of selected data sets may bedetermined by at least one predefined rule to minimize a relativestandard deviation (RSD) of intensities of the data.

In embodiments, it may be determined from a single laser pulse of aplurality of laser pulses an authentic mass of the at least one ionizedparticle and/or center of mass of the at least one ionized particle withoptimal accuracy and/or reproducibility.

Example FIG. 5 illustrates a MALDI plate where a spot on a sample plateis irradiated by a laser pulse, in accordance with embodiments. Eachtime a laser pulse is fired on a spot, a spectrum of peaks may becreated (e.g., as shown in example FIGS. 9-12). Due to non-homogeneousnature of drying sample and matrix mixture on a sample plate,intensities may vary depending on a spot hit by a laser beam. Measuredmass values and corresponding intensities may fluctuate with shots.

Example FIG. 6 is a MALDI-TOF MS hardware diagram, in accordance withembodiments. Different types of detectors 613 are available, asappreciated by those of ordinary skill in the art. A MALDI-TOF MS systemmay exploit the fact that all ions 615 a-c accelerated in the sameelectric field 605 may have the same or substantially the same kineticenergy. After leaving the electric field 605 (e.g. generated byelectrodes) ions 615 a-c may enter a field-free section and/or flighttube 603. Flight tube 603 may have a predetermined length 611. Ions 615a-c have different speeds depending on their mass. Large ions 615 a maytake more time to traverse the flight tube than smaller ions 615 c.

The matrix 607 containing a sample may be irradiated by a laser 601.Both the sample molecules on the matrix 607 may be vaporized. As thematrix 607 absorbs the laser 601 and the sample becomes ionized, some ofthat energy is passed to the sample molecules and a number of the samplemolecules become ionized 615 a-c. Voltage may be applied to electrodesin a chamber containing the matrix 607, drawing the ionized molecules615 a-c to the mass spectrometer tube 603 and ultimately to detector613.

An electrostatic field along the tube 603 of the spectrometer causes theionized molecules 615 a-e to fly down the length of the tube 603. The“time of flight” (TOF) is the time it takes the ions 615 a-c to reachthe detector 613 at the end of the tube 603 and depends on itsmass/charge ratio (m/z) of the ionized particles 615 a-c. The recordedtime is converted by the spectrometer and is reported as an m/z ratio,where m is the mass of the ion in Daltons, and z is the ions' charge.

Example FIG. 7 illustrates a laser pulse applied to sample in MALDIchamber, in accordance with embodiments. The sample 709 on substrate 707is irradiated by laser beam 701. In embodiments, laser beam 701 is UVlight. In embodiments, laser beam 701 is projected onto the sample 709at approximately a 30 degree angle, although all other angles may beused. In embodiments, ionized particles 715 a-e are produced as a resultof the laser beam 701 and drawn away from the sample.

Example FIG. 8 illustrates an ionization of matrix and sample particles807 as a result of laser beam 801, in accordance with embodiments. Thesample 807 may be irradiated by laser beam 801. The sample molecules 807are vaporized into ionized particles (e.g. ionized particles 815 or817). As the sample 807 absorbs the laser beam 801 and portions ofsample 807 become ionized, some of that energy is passed to the ionizedparticles 815 or 817. Voltage is applied to electrodes 803 and 805,drawing 819 the ionized molecules 815 or 817 to the mass spectrometertube (e.g. tube 603 of FIG. 6).

Example FIG. 9 illustrates a MALDI TOF mass spectra of ZnO. A series ofpeaks were observed in the m/z range of 500 to 2000. Major peaks wereobserved at 576 m/z corresponding to Zn₆O₁₂ ⁺ and at 868 m/zcorresponding to Zn₁₀O₁₃ ⁺. Clusters of Zn₁₄O₁₄ ⁺ and Zn₁₉O₁₇ ⁺ werealso observed as minor peaks. The data output from a detector may berepresented as a spectrum (e.g. as shown in example FIGS. 9-12), whichmay be a list of peaks that represent a list of particular moleculescombined in the original sample.

Example FIG. 10 illustrates enlarged mass spectra of FIG. 9 around 576m/z, in accordance with embodiments. It can be seen that the peak at 576m/z actually consists of multiple small peaks separated from each other.These multiple small peaks may have different m/z peaks due toattributes of the ionized particles. In embodiments, a detectorindependently measures to isolate variations in attributes of each ofthe ionized particles, which may be represented by the multiple peaks inFIG. 11. In embodiments, the attributes of the ionized particles mayinclude an acceleration efficiency of each of the ionized particlesthrough electrodes. In embodiments, the attributes of each of theionized particles may include delays in the ionized particles enteringthe flight tube. In embodiments, the attributes of the ionized particlesmay include variations of path of flight of the ionized particles insidethe flight tube.

Example FIG. 11 illustrates an example MALDI-TOF mass spectra of asample including both Zn and Au, in accordance with embodiments.Accordingly, important information can be lost (e.g. Au is not detected)since the Au peaks may be indistinguishable from the Zn peaks. Forexample, in medical diagnosis applications, an opportunity to diagnose adisease may be lost due to spread peaks of two different types ofmolecules that are close to each other. Such challenges may be overcome,in embodiments, by independently measuring the sample.

In embodiments, independently measuring may include measuring adistribution of the time of flight and intensity of the ionizedparticles. In embodiments, independently measuring is effectively anoise filter from noise produced in the target area upon beingirradiated by a plurality of laser pulses. In embodiments, the noisefilter minimizes data truncation of data measured by the detector. Inembodiments, the noise filter minimizes undesirable intensity effects ofdata measured by the detector. In embodiments, the independentlymeasuring may include independently measuring the time of flight and theintensity of the ionized particles for each pulse of the plurality oflaser pulses. In embodiments, the detector may independently measure tocompensate for physical variations in the sample. In embodiments, thedetector may independently measure to optimize data reproducibility. Inembodiments, the detector independently measures to maximize diagnosticaccuracy. In embodiments, the independently measuring may includeindependently measuring the time of flight and the intensity of theionized particles for each locus of the sample. In embodiments, theindependently measuring may include independently measuring the time offlight and the intensity of the particles for each well of target area.

Example FIG. 12 illustrates the peak at 515 m/z corresponds to the(AuClO)₂ cluster of FIG. 11, in accordance with embodiments. The peaksare made up of small peaks separated by one unit. Since Cl exists in twostable isotopic forms, ³⁵Cl and ³⁷Cl, this indicates the isotropicdistribution of the original sample. Although the AuCl peaks are lesspronounced than the Zn peaks, these smaller peaks of AuCl containvaluable information, in accordance with embodiments.

Embodiments relate to use of laser shot-to-shot data independently,instead of just summing up or averaging them. Embodiments may use thedistribution of each shot data for mass and intensity so that all thedata are statistically and analytically weighted, which may increase thereproducibility of data.

Embodiments relate to data manipulation to compensate for variations insample distributions. Even with an integrated sample preparation system(e.g. to minimize the human and environmental errors), there may beinconsistencies when adding up all the data from well to well (e.g.locus-to-locus) in a spot of the test plate. The added intensities mayvary with spot-to-spot due to the initial thickness of the sample loadsand structure of the points when a laser irradiates. For example, eachwell or locus may have sample material (to be tested) that isirregularly spread across its surface among a plurality of wells ormultiple locus divisions. Embodiments compensate for this inconsistencyby treating each locus or well separately and apply the correspondingdata manipulation techniques to each locus or well. Embodiments relateto shot-to-shot locus separation that may yield an effective filter fornoise from plate or MALDI equipment or matrix, which may result inminimal data truncation and/or intensity effect of masses in the data.Embodiments may optimize data reproducibility and/or diagnosticaccuracy.

In embodiments, a laser may be fired at the matrix crystals in thedried-droplet spot. The matrix absorbs the laser energy and it isdesorbed and ionized. A hot plume produced during ablation may containdifferent species (e.g. neutral and ionized matrix molecules, protonatedand deprotonated matrix molecules, matrix clusters, and/ornanodroplets). Ablated species may participate in the ionization ofanalyte.

MALDI/TOF spectra may be used for the identification of micro-organismssuch as bacteria or fungi, in accordance with embodiments. A sample maybe placed onto a target area and overlaid with matrix. The mass spectragenerated may be analyzed by dedicated software and compared with storedprofiles. Species diagnosis by this procedure may be quicker, moreaccurate, and/or cheaper depending on the application.

A biomarker is a biological molecule found in blood, other body fluids,or tissues that is a sign of a normal or abnormal process, or of acondition or disease. For example, a glycoprotein CA-152 is a biomarkerthat signals the existence of a cancer. Hence, biomarkers are oftenmeasured and evaluated to identify the presence or progress of aparticular disease or to see how well the body responds to a treatmentfor a disease or condition. Existence or a change in quantity level ofbiomarkers in proteins, peptides, lipids, glycan or metabolites can bemeasured by mass spectrometers.

Among numerous types of mass spectrometers, Matrix-Assisted LaserResorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) isan analytical tool employing a soft ionization technique. Samples areembedded in a matrix and a laser pulse is fired at the mixture. Thematrix absorbs the laser energy and the molecules of the mixture areionized. The ionized molecules are then accelerated through a part of avacuum tube by an electrical field and then fly in the rest of thechamber without fields. Time-of-flight is measured to produce themass-to-charge ratio (m/z). MALDI-TOF MS offers rapid identification ofbiomolecules such as peptides, proteins and large organic molecules withvery high accuracy and subpicomole sensitivity. MALDI-TOF MS may be usedin a laboratory environment to rapidly and accurately analyzebiomolecules and expanding its application to clinical areas such asmicroorganism detection and disease diagnosis such as cancers.

Disease diagnosis using MALDI-TOF MS in a clinical environment, however,presents several problems. One problem is poor reproducibility of themass analysis data. In particular, sample preparation process is a majorfactor affecting data reproducibility of MALDI-TOF MS, where a specifictarget material is extracted from an original sample, mixed with amatrix and then loaded onto a sample plate. Handling processes mayinevitable involve human intervention where a person manually movessamples from one processing step to another processing step and/orperforms a number of experimental processes. This makes the datasusceptible to uncontrolled external influences, which leads to poorhomogeneity or separability of a sample and a risk of samplecontamination.

Another factor affecting data reproducibility is the measurementsensitivity or measuring process of the MALDI-TOF MS system itself.While MALDI-TOF MS can analyze samples fast with high sensitivity sothat it would be an excellent tool for clinical application, it may be arelatively poor quantitative analyzer because Relative StandardDeviation (RSD) of detected signal intensities is relatively high due toits nature of ionization process using organic matrix. Even though theMALDI-TOF MS system adopts a delayed extraction technique, it may bechallenging to have all the particles of a mass get the same kineticenergy just before entering a field-free zone in the chamber. It may bean inevitable data spread source.

Other techniques may account for the inherent inconsistency of sampledistribution on the plates that the machine ionizes.

Even with an integrated sample preparation system, the sample may beinconsistently applied across the test plate upon which the MS laser isirradiated. A method to deal with this is to add up all the data fromwell-to-well (or locus-to-locus) in a spot of the test plate and/oraverage them to yield the representative data. This method may beinsufficient, because each well also has its sample irregularly spreadacross its surface. Embodiments overcome this inconsistency byconsidering each locus separately. Embodiments relate to a MALDI-TOF MSdata generation unit to increase data reproducibility. Some MALDI-TOF MSdevices use a sum or an average value of the data spotting on a specificsample spot of a plate. Embodiments include a calibration unit tocorrect the spotting data using a statistical method to increase thereproducibility of the data. For example, the data on a spot may not beuniform. The uniformity might be higher in a liquid form of the sample.However, a sample may be prepared in a solid form on a sample plate,then converted to a gaseous state to be analyzed intrinsically may causerelatively lower uniformity, which in turn may degrade reproducibilityof the mass data. Therefore, the data acquired during laser spotting onthe same spot should be carefully examined and calibrated for highreproducibility. Embodiments store data for each laser irradiation in aMALDI-TOF MS and calibrate intensities by eliminating the outliers ofthe intensity data for each mass-to-charge (m/z) peak or selecting adata set which shows the lowest RSD or utilizing the sufficientinformation from shot-to-shot data (e.g. converting to a reasonablycontinuous data distribution). Elimination may be done with an algorithmby which some percentages of the data shall be eliminated beforeaveraging or summation of the intensities, in accordance withembodiments. The percentage of elimination may be determined by at leastone predefined rule to minimize the RSD of the intensities.

Each time a laser pulse is fired on a spot, a spectrum of peaks may becreated. Due to non-homogenous nature of drying sample and matrixmixture on a sample plate, intensities may vary depending on a spot hitby a laser beam. Measured mass values and corresponding intensities mayvary with shots. In accordance with embodiments, a disease diagnosticssoftware or microorganism identification software include aspects of thefollowing algorithm: The mass values by a single pulse of laser arestored on a data storage space in MALDI-TOF MS system without adding toor averaging with the data obtained from other laser irradiations.AND/OR The stored mass and intensity data may then be analyzed and/orfiltered out depending upon the characteristics of the analysis ofdiseases. AND/OR The stored mass value may have a spread distributionfor each mass, so that the authentic mass value for each mass in a spotof a sample plate may be estimated for the analysis of diseaseidentification or microbial tests. AND/OR Since every laser shot yieldsa slightly different authentic mass value, each authentic mass may beadjusted to the corresponding standard mass value for diagnosticpurposes. AND/OR The measured intensity values are then normalized andcalibrated for each standard authentic mass. The stored intensity datafor each laser shot may then be put together into its distribution curvefor filtering out to reduce the RSD of the data. Embodiments relate tofinding authentic mass and/or center of mass in a single laser shot. Allthe particles of the same mass may drift into the field-free chamber ofMALDI-TOF MS with the same velocity, but in some circumstances maydeviate from the velocity of the authentic mass. The mass data obtainedfrom a detector may be calibrated for diagnostic and/or otherapplications to obtain standard mass by authentic mass and/or center ofmass information based on certain observations, in accordance withembodiments.

In embodiments, observations may relate to a deviation from theauthentic mass due to inherent nature of ions that can be denoted asI(j)*(m(i,j)−m(i,c)), where m(i,j) is a measured mass (converted fromtime) with an intensity I(j) and m(i,c) is the authentic mass or thecenter of mass related to all I(j). Since the intensity I_(j) (=I(j))may be related to the number of particles of the mass, m_(i) (=m(i,j)),the quantity m_(i)*I_(j) may be closer to the quantity of the specificmass m_(i), rather than m_(i) itself or I_(j) alone. The weightedaverage of the quantity m_(i)*I_(j) can be written asΣ(m_(i)*I_(j))/ΣI_(j) and may be treated as a kind of the center ofmass, m_(c), which may be equivalent to ΣI_(j)*(m_(i)−m_(c))=0, meaningthat the ion particles are distributed and equilibrated around thecenter of mass. Therefore, in embodiments, the authentic mass or thecenter of mass, m_(c), can be estimated using the intensity weightedmass formula as the definition above. In other words, m_(c), may be aweighted sum of all the masses around a specific (m/z) of interest. Thenumber of intensities for a mass/charge is selected based on theaccuracy of a MALDI-TOF MS, in accordance with embodiments describingdimensional effect and optimal spacing using deep learning techniqueand/or other techniques.

Embodiments relate to calibration of masses and intensities within asingle MALDI plate spot. Matrix solution mixed with a sample may bespotted on a MALDI plate, typically made of metal. A MALDI plate mayinclude multiple spots containing matrix solution mixed with a sample.Laser pulses may be fired multiple times at each spot. Because thesolution densities may not be uniform even within a spot and the part ofthe sample and matrix mixture after a laser irradiation may have adifferent structure from the one just before the previous laserirradiation, intensity variations for each laser irradiation may benatural and/or inevitable. Such intensity deviations within a spot of aMALDI-plate may be calibrated by a filtering algorithm for each m/z'sintensity distribution from the storing data of each laser irradiation.

Each time a laser pulse is fired on a spot of a MALDI plate, a spectrumof peaks may be created, in accordance with embodiments. For each peakof the spectrum, there may be a list of peaks acquired from each laserpulse irradiation. For example, if the irradiation is 1,000 times, thenumber of laser pulse samples for a mass-to-charge-ratio (m/z) peak maybe 1,000 under theoretically ideal circumstances.

In embodiments, the intensity weighted masses for each mass may becalibrated to a standard mass for diagnostic purposes such asmicro-organisms detection and/or cancer marker identification. Astandard mass may be a mass representing a mass bracket or mass range,where a mass bracket is a range of masses in which all the masses arethe identical mass called standard mass for the bracket.

A standardized mass-to-charge ratio (m/z) library may be created, inaccordance with embodiments. The range of a first mass bracket may bethe measuring time interval (e.g. time bin of the detection system ofMALDI-TOF MS). Since the ions with the same mass may have differentinitial velocity or velocity distribution at the entrance the field-freechamber of MALDI-TOF MS and enter the chamber entrance slit at differenttimes, some of the mass brackets may need to be merged as an identicalentity (e.g. those brackets may be assumed to be an identical massbracket). The merge guideline for a second mass bracket may be based onaverage mass accuracy (e.g. 100 ppm of a mass or SQRT(1+β*(D/L)̂2))dimensional constant described above. In the ppm example, any masswithin 100 ppm of a specific mass may be regarded as the same mass ofthat specific mass. Another merge guideline example for a second massbracket is to use a modified uniform interval for the first range andthen employ a concept of difference comparison in which a mass of abracket and another mass of the adjacent bracket are compared and mergedif the difference of two masses is within the modified uniform bracketinterval. For example, a table may be generated containing brackets withbase (m/z) ranging from 0 to 50,000 (or any relatively high number),each bracket having a range of (m/z)'s with an interval of 0.001 orother number (modified uniform interval), where machine accuracy error(in this example) is assumed to be greater than 1 ppm for 1,000 so that0.001 covers most of all machine errors in presence. If the minimumintensity of a bracket minus the maximum intensity of the bracket priorto the bracket of interest is less than the pre-set machine accuracyerror (e.g. 0.001 or second decimal points, 0.01), then those bracketsare merged into one, labeled with a median value of the merged ranges.

If there are two or more known (m/z)'s in any bracket range, suchbracket may be split into two or more sub-brackets. For example, if anexample median contains two or more known (m/z)'s in nature, then thebracket represented by that median split into two or more sub-brackets.

Embodiments relate to calibration of intensities within a single MALDIplate spot. Any m/z may be adjusted to the standardized m/z, inaccordance with embodiments. After all the acquired intensities arerearranged for the standard m/z, each standard m/z may have its own m/zrange and corresponding intensity obtained from each laser pulseirradiation. Each standard m/z may have an intensity distributioncontaining outliers of an abnormal character. For example, a parametertable of 1,000 laser irradiation may be constructed. In embodiments, arounded intensity value may be rounded down to two decimal places, ifthe machine error is 10 ppm for 1,000 Dalton of mass.

Several filtering guidelines may be employed to minimize the RSD, inaccordance with embodiments. For example, 90% of the high intensitiesmay be selected to be the intensities of (m/z) if its average RSD is theminimum at the selection level. Abundance (frequency of a range) orintensity level may be one of the candidates for a filtering guideline,in accordance with embodiments.

Embodiments relate to calibration of intensities to reduce spot-to-spotvariation. The intensities of a mass spectrum may vary from one spot toanother within a plate. Spot-to-spot variation may be reduced by scalingor normalizing the intensities according to a scale factor or anormalization factor that results in the minimum average RSD. AverageRSD may also be different depending on a method of selecting peaks.Thus, according to embodiments, an average RSD for eachscale/normalization factor and each method of selecting peaks may beobtained, and then the scale/normalization factor and the method thatcan minimize the average RSD is selected.

Higher accuracy in MALDI-TOF MS data may be achieved by implementing analgorithm which finds standard masses and highly reproducible intensitydata. Reproducibility may be measured by the Relative Standard Deviation(RSD) of a measurement data set. RSD is the standard deviation ofintensity divided by the average intensity of a MALDI-TOF mass spectrumpeak. Embodiments may reduce the RSD by minimizing errors in samplepreparation step through automatic processing flow and optimizingacquired measurement data through filtering data obtained from laserirradiations on a spot.

For example, protocols involving the ovarian cancer classification, mayhave an improved RSD (from 25% to 10%) by automating sample preparation,in accordance with embodiments.

Automation of sample preparation for MALDI-TOF MS analysis may besignificant in diagnosis of early-stage disease, such as cancer. Inembodiments, benign ovarian tumors and borderline ovarian tumors ofovarian cancer may be successfully classified when the technique ofsemi-automated sample preparation is applied. Enhancement ofreproducibility may be achieved by using a matrix-prespotted MALDI plateand an automation process, in accordance with embodiments, compared toother methods in which a matrix was mixed with serum glycan analytes andmanually loaded onto MALDI target plate. In embodiments, automatedsample preparation may be able to lower the RSD of MALDI-TOF MS data byat least approximately 10%. Consequently, screening accuracy of benigntumors and borderline tumors may be above approximately 75%, whichimplies that the classification accuracy between an early stage cancerpatient and a person without ovarian cancer may be greater than 75%, inaccordance with embodiments.

Embodiments relate to a MALDI-TOF MS data generation unit to increasedata reproducibility. Some MALDI-TOF MS devices use a sum or an averagevalue of the data spotting on a specific sample spot of a plate.Embodiments include a calibration unit to correct the spotting datausing a statistical method to increase the reproducibility of the data.For example, the data on a spot may not be uniform. The uniformity mightbe higher in a liquid form of the sample. However, a sample is preparedin a solid form on a sample plate, then converted to a gaseous state tobe analyzed intrinsically may cause relatively lower uniformity, whichin turn may degrade reproducibility of the mass data. Therefore, thedata acquired during laser spotting on the same spot should be carefullyexamined and calibrated for high reproducibility.

Embodiments store data for each laser irradiation in a MALDI-TOF MS andcalibrate intensities by eliminating the outliers of the intensity datafor each mass-to-charge (m/z) peak or selecting a data set which showsthe lowest RSD. Elimination may be done with an algorithm by which somepercentages of the data shall be eliminated before averaging orsummation of the intensities, in accordance with embodiments. Thepercentage of elimination may be determined by at least one predefinedrule to minimize the RSD of the intensities.

In accordance with embodiments, a disease diagnostics software ormicroorganism identification software include aspects of the followingalgorithm: The mass values by a single pulse of laser are stored on adata storage space in MALDI-TOF MS system without adding to or averagingwith the data obtained from other laser irradiations. AND/OR The storedmass and intensity data may then be analyzed and/or filtered outdepending upon the characteristics of the analysis of diseases. AND/ORThe stored mass value may have a spread distribution for each mass sothat the authentic mass value for each mass in a spot of a sample platemay be estimated for the analysis of disease identification or microbialtests. AND/OR Since every laser shot yields a slightly differentauthentic mass value, each authentic mass may be adjusted to thecorresponding standard mass value for diagnostic purposes. AND/OR Themeasured intensity values are then normalized and calibrated for eachstandard authentic mass. The stored intensity data for each laser shotmay then be put together into its distribution curve for filtering outto reduce the RSD of the data.

Embodiments relate to finding authentic mass and/or center of mass in asingle laser shot. All the particles of the same mass may drift into thefield-free chamber of MALDI-TOF MS with the same velocity, but in somecircumstances may deviate from the velocity of the authentic mass. Themass data obtained from a detector may be calibrated for diagnosticand/or other applications to obtain standard mass by authentic massand/or center of mass information based on certain observations, inaccordance with embodiments.

In embodiments, observations may relate to a deviation from theauthentic mass due to inherent nature of ions that can be denoted asI_(i)*(m_(i)−m_(c)), where m_(i) is a measured mass with an intensityI_(i), and m_(c) is the authentic mass or the center of mass. Since theintensity I_(i) is related to the number of particles of the mass,m_(i), the quantity I_(i)*m_(i) may be closer to the quantity of thespecific mass m_(i), rather than itself or I_(i) itself. The sum of thequantity ΣI_(i)*m_(i) can be defined as m_(c)I_(c) where I_(c) is ΣI_(i)and m_(c)=ΣI_(i)*m_(i)/ΣI_(i). This may be equivalent toΣI_(i)*(m_(i)−m_(c))=0, meaning that the ion particles are distributedand equilibrated around the center of mass. Therefore, in embodiments,the authentic mass or the center of mass, m_(c), can be estimated usingthe intensity weighted mass formula, m_(c)=ΣIi*m_(i)/ΣI_(i) as thedefinition above. In other words, m_(c) may be a weighted sum of all themasses around a specific (m/z) of interest. The number of intensitiesfor a mass/charge is selected based on the accuracy of a MALDI-TOF MS,in accordance with embodiments.

Embodiments relate to calibration of (m/z)'s and intensities within asingle MALDI plate spot. Matrix solution mixed with a sample may bespotted on a MALDI plate, typically made of metal. A MALDI plate mayinclude multiple spots containing matrix solution mixed with a sample.Laser pulses may be fired multiple times at each spot. Because thesolution densities may not be uniform even within a spot and the part ofthe sample and matrix mixture after a laser irradiation may have adifferent structure from the one just before the previous laserirradiation, intensity variations for each laser irradiation may benatural and/or inevitable. Such intensity deviations within a spot of aMALDI-plate may be calibrated by a filtering algorithm for each m/z'sintensity distribution from the storing data of each laser irradiation.

Each time a laser pulse is fired on a spot of a MALDI plate, a spectrumof peaks may be created, in accordance with embodiments. For each peakof the spectrum, there may be a list of peaks acquired from each laserpulse irradiation. For example, if the irradiation is 1,000 times, thenumber of peaks for a mass-to-charge (m/z) shall be 1,000 if all thepeaks are above noise threshold level. Those (m/z)'s of relativelyslight differences may be calibrated to the corresponding standardmass/charge (m/z).

In embodiments, the intensity weighted masses for each mass may becalibrated to a standard mass for diagnostic purposes such asmicro-organisms detection and/or cancer marker identification. Astandard mass may be a mass representing a mass bracket, where a massbracket is a range of masses in which all the masses are the identicalmass called standard mass for the bracket.

A standardized mass-to-charge ratio (m/z) library may be created, inaccordance with embodiments. The range of a first mass bracket may bethe measuring time interval (e.g. time bin of the detection system ofMALDI-TOF MS). Since the ions with the same mass may have differentinitial velocity at the entrance the field-free chamber of MALDI-TOF MS,some of the mass brackets may need to be merged as an identical entity(e.g. those brackets may be assumed to be an identical mass bracket).The merge guideline for a second mass bracket may be based on averagemass accuracy (e.g. 100 ppm of a mass). In this example, any mass within100 ppm of a specific mass may be regarded as the same mass of thatspecific mass.

Another merge guideline example for a second mass bracket is to use amodified uniform interval for the first range and then employ a conceptof difference comparison in which a mass of a bracket and another massof the adjacent bracket are compared and merged if the difference of twomasses is within the modified uniform bucket interval. For example, atable may be generated containing brackets with base (m/z) ranging from0 to 50,000 (or any relatively high number), each bracket having a rangeof (m/z)'s with an interval of 0.001 (modified uniform interval), wheremachine accuracy error (in this example) is assumed to be greater than 1ppm for 1,000 so that 0.001 covers most of all machine errors inpresence. If the minimum intensity of a bracket minus the maximumintensity of the bracket prior to the bracket of interest is less thanthe pre-set machine accuracy error (e.g. 0.001 or second decimal points,0.01), then those brackets are merged into one, labeled with a medianvalue of the merged ranges.

If there are two or more known (m/z)'s in any bracket range, suchbracket may be split into two or more sub-brackets. For example, if anexample median contains two or more known (m/z)'s in nature, then thebracket represented by that median split into two or more sub-brackets.

Embodiments relate to calibration of intensities within a single MALDIplate spot. Any m/z may be adjusted to the standardized m/z, inaccordance with embodiments. After all the acquired intensities arerearranged for the standard m/z, each standard m/z may have its own m/zrange and corresponding intensity obtained from each laser pulseirradiation. Each standard m/z may have an intensity distributioncontaining outliers of an abnormal character. For example, a parametertable of 1,000 laser irradiation can be constructed. In embodiments, arounded intensity value may be rounded down to two decimal places, ifthe machine error is 10 ppm for 1,000 of m/z.

Several filtering guidelines may be employed to minimize the RSD, inaccordance with embodiments. For example, 90% of the high intensitiesmay be selected to be the intensities of (m/z) if its average RSD is theminimum. Abundance (frequency of a range) or intensity level may be oneof the candidates for a filtering guideline, in accordance withembodiments.

Embodiments relate to calibration of intensities to reduce spot-to-spotvariation. The intensities of a mass spectrum may vary from one spot toanother within a plate. Spot-to-spot variation may be reduced by scalingor normalizing the intensities according to a scale factor or anormalization factor that results in the minimum average RSD. AverageRSD may also be different depending on a method of selecting peaks.Thus, according to embodiments, an average RSD for eachscale/normalization factor and each method of selecting peaks may beobtained, and then the scale/normalization factor and the method thatcan minimize the average RSD is selected.

In embodiments, with an automated procedure of sample preparation and/orcalibration methods, there may be significant enhance thereproducibility of the MALDI-TOF MS for a diagnostic purpose, such acancer diagnosis.

It will be obvious and apparent to those skilled in the art that variousmodifications and variations can be made in the embodiments disclosed.This, it is intended that the disclosed embodiments cover the obviousand apparent modifications and variations, provided that they are withinthe scope of the appended claims and their equivalents.

What is claimed is:
 1. An apparatus: a laser configured to irradiate aplurality of laser pulses on a target area to ionize a sample placed inthe target area into at least one ionized particle; at least oneelectrode at a first end of a flight tube configured to accelerate theat least one ionized particle into the flight tube; and a detector at asecond opposite end of the flight tube which independently measures atime of flight of the at least one ionized particle through the flighttube and an intensity of the at least one ionized particle.
 2. Theapparatus of claim 1, wherein said independently measures comprisesmeasuring a distribution of the time of flight and intensity of the atleast one ionized particle.
 3. The apparatus of claim 2, wherein saidindependently measures is effectively a noise filter from noise producedin the target area upon being irradiated by a plurality of laser pulses.4. The apparatus of claim 3, wherein the noise filter minimizes datatruncation of data measured by the detector.
 5. The apparatus of claim3, wherein the noise filter minimizes undesirable intensity effects ofdata measured by the detector.
 6. The apparatus of claim 1, wherein saidindependently measures comprises independently measuring the time offlight and the intensity of the at least one ionized particle for eachpulse of the plurality of laser pulses.
 7. The apparatus of claim 1,wherein the detector independently measures to isolate variations inattributes of each of the ionized particles.
 8. The apparatus of claim7, wherein the attributes of each of the ionized particles comprises anacceleration efficiency of each of the ionized particles through the atleast one electrode.
 9. The apparatus of claim 7, which the attributesof each of the ionized particles comprises delays in at least one of theplurality of ionized particles entering the flight tube.
 10. Theapparatus of claim 7, wherein the attributes of each of the ionizedparticles comprises variations of path of flight of at least one of theplurality of ionized particles inside the flight tube.
 11. The apparatusof claim 1, wherein the detector independently measures to compensatefor physical variations in the sample.
 12. The apparatus of claim 1,wherein the detector independently measures to optimize datareproducibility.
 13. The apparatus of claim 1, wherein the detectorindependently measures to maximize diagnostic accuracy.
 14. Theapparatus of claim 1, wherein the independently measures comprisesindependently measuring the time of flight and the intensity of the atleast one ionized particle for each locus of the sample.
 15. Theapparatus of claim 14, wherein said independently measures comprisesindependently measuring the time of flight and the intensity of the atleast one ionized particle for each well of target area.
 16. Theapparatus of claim 1, wherein: the detector outputs data whichrepresents the independent measurements; and the data output from thedetector is compared to a reference library of mass spectrometerreference data.
 17. The apparatus of claim 16, wherein the samplereference library is stored in at least one of a storage device, aMatrix-Assisted Laser Desorption/Ionization Time-of-Flight MassSpectrometer (MALDI-TOF MS), a data storage device in the apparatus, adata storage device outside the apparatus, a data storage device incommunication with the apparatus through a network, a cloud storagesystem, or a data storage device in communication with the apparatusthrough an internet connection.
 18. The apparatus of claim 16, whereinthe data output from the detector is at least one of manipulated oranalyzed prior to being compared to the reference library.
 19. Theapparatus of claim 18, wherein the at least one of manipulated oranalyzed comprises implementation of at least one of artificialintelligence or a deep learning algorithm.
 20. The apparatus of claim18, wherein the at least one of manipulated or analyzed data comprisescalibrated data produced by statistically analyzing the data outputfront the detector.
 21. The apparatus of claim 20, comprising acalibration unit configured to statistically calibrate the data outputfrom the detector to maximize the reproducibility of the data.
 22. Theapparatus of claim 21, wherein the calibrating unit is configured tocalibrate intensities of the data output from the detector byeliminating outliers of the intensity data for each mass-to-charge (m/z)peak associated with the at least one ionized particle.
 23. Theapparatus of claim 21, wherein the calibrating unit is configured toselect at least one data set comprising the data output from thedetector which shows a relative standard deviation (RSD) satisfying atriggering threshold.
 24. The apparatus of claim 23, wherein thetriggering threshold is at least one of: a predetermined threshold; adynamic threshold; a statistically determined threshold; a real-timevarying threshold; a threshold determined by artificial intelligence; ora threshold determined by a deep learning algorithm.
 25. The apparatusof claim 22, wherein the calibrating unit is configured to select atleast one data set comprising the data output from the detectorutilizing continuous data distribution analysis after the plurality oflaser pulses.
 26. The apparatus of claim 25, wherein the select at leastone data set comprises using an algorithm by which a percentage of thedata is eliminated.
 27. The apparatus of claim 26, wherein the algorithmis performed before combining a plurality of data sets of the at leastone data set.
 28. The apparatus of claim 27, wherein the combiningcomprises at least one of averaging or summing of the intensities of theplurality of data sets.
 29. The apparatus of claim 26, wherein thepercentage is determined by at least one predefined rule to minimize arelative standard deviation (RSD) of intensities of the data.
 30. Theapparatus of claim 1, wherein: the sample comprises molecules; andcharacteristic information of the source comprises a biological analysisinformation of the source.
 31. The apparatus of claim 30, wherein thebiological analysis information is a medical diagnosis of at least oneof a human being, an animal, a plant, or a living organism.
 32. Theapparatus of claim 1, wherein the apparatus is a Matrix-Assisted LaserDesorption/Ionization Time-of-Flight Mass Spectrometer (MALDI-TOF MS).33. The apparatus of claim 1, wherein the apparatus determines from asingle laser pulse of the plurality of laser pulses at least one of anauthentic mass of the at least one ionized particle or center of mass ofthe at least one ionized particle.
 34. A method of identifying abiomaterial using a Matrix-Assisted Laser Desorption IonizationTime-of-Flight Mass Spectrometry (MALDI-TOF MS), wherein the methodcomprising: irradiating a plurality of laser pulses on a target area toionize a sample placed in the target area into at least one ionizedparticle; accelerating the at least one ionized particle into a firstend of a flight tube; and independently detecting at a second oppositeend of the flight tube a time of flight of the at least one ionizedparticle through the flight tube and an intensity of the at least oneionized particle.
 35. A computer program product, comprising a computerreadable hardware storage device having computer readable program codestored therein, said program code containing instructions executable byone or more processors of a computer system to implement a method ofassessing damage to an object, said method comprising: irradiating aplurality of laser pulses on a target area to ionize a sample placed inthe target area into at least one ionized particle; accelerating the atleast one ionized particle into a first end of a flight tube; andindependently detecting at a second opposite end of the flight tube atime of flight of the at least one ionized particle through the flighttube and intensity of the at least one ionized particle.